Project 1:Previous studies have shown that the Notch1 and TGF-&#946; signaling pathways are mutually re-enforcing. Given recent evident that regulatory T cell (Treg) effector function is mediated by TGF-&#946; signaling, we investigated if Notch1 signaling also participated in Treg effector function. Initial studies showed that Notch1 ligands, particularly Jagged1, is present on Tregs and that, indeed, blockade of Notch1 signaling with a anti-Jagged1 or a blocking anti-Notch1 antibody inhibits Treg suppressor function in vitro. We then showed that a signaling component generated by Notch1 activation (NICD) physically interacts with a signaling component generated by TGF-&#946; signaling (pSmad3). Furthermore, this interaction has functional downstream effects since over-expression of NICD facilitates pSmad3 translocation to the nucleus and enhances pSmad3 transcriptional activity of a Smad sensitive promoter linked to a luciferase reporter. Finally, we showed that blockade of TGF-&#946; signaling and Notch signaling did not have additive inhibitory effects on Treg suppressor function. These results are consistent with the conclusion that Notch1 signaling enhances and facilitates TGF-&#946;-mediated effector function of Tregs.[unreadable] Project 2: Recent studies have shown that TGF-&#946; together with IL-6 induce the differentiation of IL-17-producing T cells (Th17) T cells. We therefore examined if CD4+CD25+Foxp3+ regulatory T cells (Tregs), i.e., cells previously shown to produce TGF-&#946;, serve as Th17 inducers. We found that upon activation purified CD25+ T cells (or sorted GFP+ T cells obtained from Foxp3-GFP knock-in mice) produce high amount of soluble TGF-&#946; and when cultured with CD4+CD25-Foxp3- T cells in the presence of IL-6 induce the latter to differentiate into Th17 cells. Perhaps more importantly, upon activation, CD4+CD25+Foxp3+(GFP+) T cells themselves differentiate into Th17 cells in the presence of IL-6 (and in the absence of exogenous TGF-&#946;). These results indicate that CD4+CD25+Foxp3+ regulatory T cells can function as inducers of Th17 cells and can differentiate into Th17 cells. They thus have important implications to our understanding of regulatory T cell function and their possible therapeutic use.